Several publications and patent documents are cited throughout this application to better define the state of the art to which this invention pertains. Each of the foregoing citations is incorporated by reference herein.
Yeast two-hybrid systems (Chien et al. 1991; Fields and Song 1989; Gyuris et al. 1993; Vojtek et al. 1993) are standard tools used to identify novel protein-protein interactions and to perform structure-function analysis on previously defined protein-protein interactions. Such systems are effective with a substantial fraction of eukaryotic proteins and have played an important role in high throughput proteomic analyses aimed at establishing sets of interacting proteins (e.g. (Giot et al. 2003; Ito et al. 2000; Li et al. 2004; Uetz et al. 2000). In order to increase the power of a two-hybrid approach to identify and analyze protein interactions in high throughput applications, one approach has been to translate the basic components of the yeast two-hybrid system to a bacterial host organism (Dove et al. 1997; Joung et al. 2000). To date, the relative effectiveness of protein interaction detection in bacterial and yeast backgrounds has not been directly compared. However, there are a number of reasons to anticipate that differences might be observed. As yeast are eukaryotes, eukaryotic proteins used as “baits” in two-hybrid screens may be more likely to be appropriately folded and post-translationally modified in yeast than in bacteria, thereby increasing their chances of identifying physiological partners. However, certain proteins can be problematic as baits in the yeast two-hybrid system; for example, proteins that are normally excluded from the nucleus in eukaryotes, that are potentially sequestered via interaction with an abundant partner evolutionarily conserved in yeast, or that stimulate transcription in yeast (i.e.—that “autoactivate”). All of these potential issues would be expected to be less problematic in the bacterial two-hybrid system. To maximize chances of obtaining all relevant interactors for a protein of interest, it would be desirable to have the capability to rapidly test a given bait in both yeast and bacterial milieus.